Encapsulation of sperm for artificial insemination

ABSTRACT

Sperm are encapsulated in a nontoxic polymer which is freely flowing at body temperature and a gel or solid at temperatures of storage and transfer. On delivery to the reproductive tract, the polymer microcapsule liquifies and the sperm are released.

FIELD OF INVENTION

The present invention relates generally to the storage, transport,maintenance and insemination of viable sperm.

BACKGROUND OF INVENTION

For breeding of animals, maximum fertilization is dependent upon preciseinsemination of sufficient numbers of healthy, viable sperm at theappropriate times. The decrease in fertilization due to poor timing ofinsemination may be a result of either a loss of sperm viability or areduction in numbers of viable sperm available during the fertile lifeof the ovum. It is desirable to improve conception rates in artificiallybred animals through the prolongation of viable sperm retention andrelease in the female reproductive tract.

Studies have already been undertaken by several laboratories to evaluatethe efficiency of microencapsulation of spermatozoa as a means ofenhancing sperm retention in the female following artificialinsemination. Microencapsulation is based on the fact that large lossesof sperm occur from and within the female during insemination,especially after the deposition of previously cryopreserved spermatozoa.The major causes of sperm disappearance appear to be leucocyticphagocytosis (engulfment of sperm by white blood cells which invade theuterus at the time of heat), and retrograde removal (expulsion of spermfrom the uterus back through the cervix).

It has been postulated that microencapsulation will protect sperm fromwhite blood cells and retrograde removal, and with appropriate capsuleconstruction, could provide a prolonged release of sufficient numbers ofsperm at a preprogrammed time to allow fertilization in a singleinsemination. Also, because sperm need not be cryopreserved prior toinsemination, normal loss occurring as a consequence of thawing from thefrozen state would not be a factor. This would allow for greater numbersof viable sperm to be deposited.

"Microencapsulation", as used herein, is defined as a process wherebysmall living cells are completely surrounded and enclosed by relativelyinert materials to form a microcapsule. Microcapsules range in diameterfrom approximately 0.2 microns to several millimeters.

Microcapsules have been provided with both impermeable and semipermeablemembranes, depending on the composition of the enclosed material and theuse intended. The mechanism(s) for release were associated withleaching, erosion, rupture, or other such actions, depending uponconstruction of the capsule wall.

Nebel, et al., J. Anim. Sci., 60: 1631 (June 1985) reported themicroencapsulation of bovine spermatozoa. Their microcapsules differedfrom those of the present invention in that they were not designed toassume a liquid state at a particular temperature. Sperm cells weresuspended in a sodium alignate and fine droplets of this suspension wereproduced using a syringe pump extrusion technique. The droplets werecollected in a calcium chloride solution which results in an immediategellation of the droplets, thus producing a shape-retaining highviscosity mold for the microcapsules. A semi-permeable membrane wasapplied to the droplets by suspending them in a solution of polylysine,thus forming the microcapsules. The gelled suspension of sperm insidethe capsules was then liquified by exposing the capsules to a solutionof sodium citrate, rather than by raising the temperature to bodytemperature. Once a sperm suspension in the capsule was liquified, thesperm resumed the mobility which had been arrested temporarily bygellation.

However, the sperm showed diminished motile life following suchencapsulation and total loss of fertilizability. Further studiesindicated that when biodegradable microcapsules were used, the retentionof the sperm was poor. The biodegradable microcapsules were also proneto early rupture and retrograde removal.

Drug delivery means which are solid or gel-like at room temperature andliquid at body temperature are known. These do not allow for providingnutrients and oxygen to living cells to maintain their viability. Also,they do not provide or require a sharp phase transition with respect totemperature.

The present invention contemplates the encapsulation of living, viablesperm such that the sperm's motility, viability, retention and releasein the female reproductive tract is not significantly impaired.

SUMMARY OF THE INVENTION

The invention contemplates the storage, transportation, and maintenanceof sperm cells in a viable state for relatively prolonged periods oftime, and their delivery in viable form to the female reproductivetract. Purified sperm are encapsulated in a nontoxic, hydrophilicpolymer, the polymer being permeable to gases and small molecules (5000daltons or less) and relatively impermeable to macromolecules. Thepolymer must assume a flowable liquid state at the body temperature ofthe recipient (typically 37° C.) and exist in a solid or semi-solid gelstate at temperatures below such body temperature.

The polymer, is preferably a reversible hydrogel of a water insoluble,hydrophilic, polyurethane polymer.

An ideal encapsulating polymer would have the following characteristics:

[1] nontoxicity, the polymer must have a chemical composition that willbe inert to cells and be unable to elicit an immune response when placedin situ;

[2] programmability, the polymer must be able to change its physicalstate as a function of an environmental factor such as temperature, pH,specific ion concentration. The desired change in physical state is froma waterlike fluid to-viscous fluid-to a solid gel (and vice versa) overa predetermined period of time, and under a predetermined environmentalcondition, such as temperature;

[3] matrix formation, the polymer must be composed nearly entirely ofwater, have a predetermined porosity, freely exchange gas and moleculesof low molecular weight (metabolites) and be able to maintain a constantpH throughout the matrix;

4] cryopreservability, the polymer when fully formed with cells enclosedshould be able to be prepared for routine cryopreservation in liquidnitrogen without irreversible alteration in structure or physicalproperties and without significant damage to cellular contents; and

[5] deliverability, the polymer must be able to get prepared in such aform as to make its delivery or deposition within the reproductive tractrelatively fast and simple.

The microcapsule must be permeable to small molecules so that nutrients,including oxygen, may reach the sperm, and so waste products, includingcarbon dioxide, may be eliminated. Preferably, the polymer provides amolecular weight cutoff of about 5,000 daltons.

While the encapsulated sperm may be maintained for a time at roomtemperature, in order to reduce the rate of build-up of potentiallytoxic metabolites within the microcapsule, the encapsulated sperm arepreferably kept at lower temperatures, such as 4° C.

In general, a more solid gel is preferable, since it will more narrowlyconstrain the movement of the sperm within the microcapsule. Byarresting the movement of the sperm, its metabilic activity is reduced,with the aforestated advantages.

The microcapsule must be as free as possible of substances toxic toeither the sperm or the recipient animal. In this regard, it isimportant that the polymer be washed thoroughly to remove toxicchemicals, such as the diisocyanate used in the preparation of apolyurethane polymer.

Success in fertilization is very dependent upon the pH in the uterus. Itis undersirable to deliver sperm to the uterus in a manner whichadversely affects pH. The culture medium in which the sperm reside whenencapsulated should have a pH in the desired range 7.2 to 7.6.

It is desirable that the microcapsule be translucent so that thecondition of the sperm is observable by phase contrast or differentialinterference contrast microscopy.

As used herein, the term "animal" is intended to include humans. .Iadd.A"warm-blooded animal" is one whose reproductive tract temperature isnormally substantially in excess of room temperature (22° C.). .Iaddend.

While the body temperature of animals typically selected for artificialinsemination is generally 37° C., it is possible that particular speciesor individuals will evince body temperatures markedly below or abovethis figure. In this event, it is within the contemplation of thisinvention to modify the composition of the encapsulating polymer so thatits state transition occurs at a higher or lower temperature.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, sperm are encapsulated in porous orsemipermeable hydrophilic polymer that is relatively inert to the cellsit surrounds and to the site into which it is deposited, such that animmune response does not take place, In addition, the hydrophilicpolymer is designed to maintain a constant pH throughout its matrix, toallow free passage of low molecular weight molecules such as carbondioxide, oxygen, glucose and amino acids through its pores and to form aprotective barrier against cells or macromolecules, or both, which couldengulf, attack, or react with the encapsulated sperm. The polymerpreferably provides a sufficient protective environment around the spermso that they are able to survive at temperatures as low as 4° C.

Release from the encapsulate is fast and simple. Merely by putting thecapsule into an environment maintained at approximately 37° C., thepolymer progressively becomes liquid, thereby freeing the sperm to swinaway.

For the purposes of this invention a variety of thermally reversiblehydrophilic polymers are satisfactory. In the preferred embodiment ofthe invention, the polymer is a thermally reversible hydrogen, being aflowable liquid (sol) at the body temperature of the recipient,typically about 37° C. but a solid semigel at the lower temperaturesexperienced during storage and transportation, typically about 15° orless. The polymer must be water insoluble at normal ambient temperaturesbut still absorb water. The preferred polymers are thepolyurethane-polyether polymers that are readily prepared by thereaction of long chain polyoxyethylene diols or glycols withpolyfunctional isocyanates. The polymers may be modified by the additionof uarrcus functional groups, such as lactone groups, carboxylate groupsand/or hydroxyl groups in the polymer backbone. Such modifications canbe used to tailor the characteristics of the polymer, for example, itspermeability and translucency. In general, the polymers are prepared bypreparing a homogeneous melt or mixture of the long chainpolyoxyethylene diol (polyether and/or polyester) and a low molecularweight glycol and reacting the melt or mixture with a diisocyanate.

The preparation of the water insoluble, hydrogel forming, hydrophilicpolyurethane and modified hydrophilic polyurethane polymers is disclosedin numerous patents such as, for example, U.S. Pat. Nos. 3,822,238;3,975,350; 4,156,066; 4,156,067 and 4,255,550; the disclosures of whichare incorporated herein by reference. Since the preparation of thesepolymers form no part of the present invention, only the preparation ofrepresentative polymers of this class which are satisfactory for thepurposes of the present invention are described below.

A convenient source of the polyether moiety of the preferred polymersare the various grades of Carbowax (R) polyoxyethylene glycols (UnionCarbide Corporation, Danbury, Conn.) and the Pluronic (R) blockcopolymers of ethylene oxide and propylene glycols (WyndotteCorporation, Parsippany, N.J.) A convenient source of a satisfactorydiisocyanate is methylene bis-cyclo-hexyl-4,4'-diisocyanate (Desmodur Wof Mobay Chemical Corporation, Pittsburgh, Pa.). Catalysts useful informing the polymer include dibutyl tin dilaurate and stannous octoate(T12 and T9, respectively, of Metal and Thermite Company, Rahway, N.J.).

To prepare the encapsulated sperm, the sperm are suspended in thedesired aqueous culture medium, and are encapsulated in the polymerhydrogel by slowly mixing the suspension with the hydrogel in flowableliquid state at normal animal temperatures and the mixture chilled,typically over a period of 0.5 to 18.5 hours, to a temperaturesufficiently low to allow the hydrogel to revert to or set into itssolid gel state. For example, the mixture may be formed at a temperatureof around 37°-39° C. and the mixture gradually chilled over a period ofminutes to hours, depending on the source (species) of sperm, to normalroom temperature, 22° C., but preferably to a lower temperature such as4°-5° C. The encapsulated sperm are completely immobilized in thepolymer and may be stored and transported at the lower temperatures tothe site of use. The microcapsule preferably provides a gaseousatmosphere of 5% O₂, 5% CO₂, and 90% N₂ and a pH of 7.25-7.35. This maybe accomplished by equilibrating the polymer with the desired atmosphereand storing it in a sealed container in a medium buffered to proper pH.In preparing the polymer hydrogel, the water or aqueous phase preferablyis comprised of culture medium, preferably buffered to maintain a pH of7.37-7.42, and containing nutrients to sustain minimal metabolic needsof the sperm. A typical aqueous culture medium is Ham's F10 solutionwith half the normal glucose concentration, but the invention herein isnot limited to any particular culture medium. Preferably, calciumlactate is added to the culture medium shortly before the sperm areencapsulated by polymerization.

Upon introduction of the capsule of sperm by standard artificialinsemination methods, the temperature rises to the uterine bodytemperature of the recipent, whereby the hydrogel progressively revertsto its flowably liquid state and releases the sperm over a predeterminedlength of time.

EXAMPLE 1 Determining the Suitablity of a Polymer for Gamete Storage

In considering the suitability of various polymers for gamet storage,several physical conditions were thought desirable: (1) the polymer mustallow free passage of metabolites, small molecules, and gasses tomaintain uniform chemical composition and pH; (2) the polymer mustundergo phase transition at a predictable rate and time; and (3) thepolymer must have a fairly uniform porosity preferably not greater than1 micron.

PERMEABILITY

Permeability was determined by incubating polymer cubes from 1 to 9%concentration in medium containing S³⁵ -L-methionine. At 5 minuteintervals polymer was removed and a 0.5 mm core sample was obtained.Radioactivity in the core was determined by liquid scintillationspectroscopy. The results of a typical run with three concentrations ofpolymer are presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        ACCUMULATION OF 35S METHIONINE                                                IN CENTER OF POLYMER                                                                      POLYMER CONCENTRATION                                                         1%       5%     9%                                                              TOTAL RADIACTIVITY                                              TIME (MINUTES)                                                                              (CPM × 1 MILLION)                                         ______________________________________                                         1             2.8        3.5    3.2                                           5            12.7       16.8   14.7                                          10            13.9       17.4   15.2                                          15            12.4       16.8   13.3                                          30            17.2       15.1   14.7                                          45            16.5       15.8   15.3                                          ______________________________________                                    

These results demonstrate that concentration of polymer has no visibleinfluence on rate of accumulation of radioactivity in center of a 5 mmcube and that substantial equilibrium with the environment requires lessthan 5 minutes

Maintenance of pH

Maintaining a constant and uniform pH throughout the polymer isabsolutely required for survival of mammalian gametes. To test theability of polymer to assume pH, polymer was gelled around a micro pHelectrode at a adminal pH of 7. The polymer-electrode assembly was thenplaced in culture medium made at pH ranging from 5.5 to 8.5 (ambientpH). The rate of change of pH from a nominal pH 7 to ambient pH wasmeasured. In polymer concentrations of 1,4 and 9%, pH change to ambientat the core occurred within 45 seconds. This result demonstrates thatthe entire polymer can respond rapidly to changes in environmental pH.

Preferably, the polymer used for entrapment of spermatozoa had anaverage pore size of no more than one micron, although porosities up to4 microns in diameter should be acceptable. To determine approximate,relative porosity as a function of polymer concentration, blocks ofpolymer were quick frozen in liquid nitrogen and sectioned in anulramicrotome maintained at liquid nitrogen temperature. The frozen thinsections were mounted on an electron microscope grid. Under standardconditions used to prevent artifactial shrinkage of specimens, the gridswere critical point dried, whereby frozen water is replaced by liquidCO₂. The specimens were examined with a million volt electron microscopeand approximate pore size distributions measured. The results are shownin Table 2.

                  TABLE 2                                                         ______________________________________                                        POLYMER                                                                       CONCENTRATION  APPROX. PORE SIZE RANGE                                        ______________________________________                                        1%               5-8 microns                                                  2%               5-8 microns                                                  3%               2-4 microns                                                  4%               1-3 microns                                                  4.5%             1-2 microns                                                  5.5%           0.5-1.5 microns                                                6%             0.3-1.5 microns                                                8%             less than 0.5 microns                                          ______________________________________                                    

Phase Transition

Polymer at concentrations of 4% and 8 % (W/V) were utilized for studiespolymers of the rate of transition to complete gelation [S] orliquification [L]. The results for these studies are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        TIMING OF PHASE TRANSITION                                                                       4%      8%                                                 ______________________________________                                        SOLID TO LIQUID                                                               TIME (4-37C)                                                                  (Minutes)                                                                      5                   S         S                                              10                   S         S                                              15                   S         S                                              30                   S         S                                              45                   S/L       S                                              75                   L         S/L                                            120                  L         L                                              LIQUID TO SOLID (37-4C)                                                        5                   L         S                                              10                   S/L       S                                              15                   S         S                                              ______________________________________                                    

These results demonstrate that the transition from solid to liquidoccurs much more slowly than the transition from liquid to solid.

EXAMPLE 2 Encapsulation of Spermatozoa

Polymers used in our studies include RL39-41; RL39-110; RL39-111;RL39-114; RL39-115; RL39-117; and RL39-118 (Tyndale Plains-Hunter Ltd.)Polymer concentrations ranged from 1.5 to 11.5%. For these trials,polymers RL39-115, 117 and 118 demonstrated a rate of cooling to finalhardness sufficient to maintain at least 60% of the encapsulatedspermatozoa in a viable state. Sperm used in this study were from mouse,bull and human sources.

It was demonstrated that spermatozoa could be maintained in the polymermicrocapsule for as many as 10 days at 110° C., and upon return to aliquid phase during a 12 hour incubation at 37° C., motility resumed in80 percent of the cells. Subsequently, it was demonstrated by in vitrofertilization that some of these sperm retained the ability to fertilizean oocyte. Similar motility observations were made with humanspermatozoa but in vitro fertilization was not attempted.

The following protocol was employed with mouse sperm; 10.7 gms ofpolymer RL-39-115 was added to 50 ml of culture grade water. The polymerwas fully hydrated and sterilized by high temperature and pressure.Concentrated medium was added, the polymer stirred vigorously andgradually cooled to 38° C. The final polymer concentration was 5.35%. At38° C., 200 million sperm contained in 80 μl were added to the polymersolution and stirred vigorously for 5 minutes. The polymer-spermdispersion was poured into molds of various shapes (3×35 mm cylinders;5×15×40 rectangles), and the molds placed in a gas tight incubator at37° C. containing 90% N₂ ; 5% CO₂ ; 5% O₂. This incubated chamber wascooled at a controlled rate ranging from 0.5° C./min. to 15° C./min.Final temperatures attained ranged from 15° C. to 3° C. Observations ofsperm activity were made by means of a time-lapse video system attachedto a Nikon Diaphot microscope equipped with differential interferencecontrast optics (NORMARSKI) and a Dage television camera. Observationswere made at 5 minute intervals during cooling and continuously duringreheating. The microscope stage was enclosed by a small chamber thatprecisely maintained temperature and held a specific gaseous atmosphere(90% N₂ 5% O₂ 5% CO₂) during all operations. Reheating occurred over aperiod ranging from 5 minutes to 18 hours.

Depending upon the rates of heating and the species, movements ofspermatozoa generally preceded total liqufication of polymer. Mousesperm (as well as human and bovine) prepared in this manner had noapparent kinetic activity when microcapsule temperature in polymerreached 7° C., at which time the polymer was fully gelled. Uponreheating, activity was first observed at 20° C. at which time thepolymer was in a semi-gel state. Sperm activity did not return uniformlythroughout the capsule but rather first appeared at the peripheralregions of the capsule and lastly at the more interior regions. Kineticactivity was a function of liquification, the process being morecomplete at the periphery and proceeding to the interior with time.Sperm obtained from the reheated capsule were mixed with fresh medium,centrifuged at 500 g for 10 min., and then allowed to "swim" away fromthe centrifugate obtained from centrifugation. Some of the mouse andbovine sperm so obtained were shown to be capable of fertilizing asuitably mature oocyte in vitro.

Human sperm was prepared in same way and tested in same fashion.Viability was assessed by morphology, general motility and theappearance of active foward progressive motility. No fertilizations wereattempted with human spermatozoa.

EXAMPLE 3 Encapsulation of Bovine Spermatozoa

Polymer at a concentration of 3.8% (dry weight/liquid) in culture mediumcontaining salts, lactate, pyruvate but not glucose or fructose wasused. Bovine in spermatozoa encapsulated in the polymer were preferablycooled from 37° C. to 10° C. over a period of at least 6 hours, a muchslower rate than that found to be effective for encapsulated murine orhuman spermatozoa. The preferred rate of temperature increase forrestoring the motility of bovine spermatozoa (gel to liquidtransformation) is also longer than for encapsulation of spermatozoa ofthe other two species (12-16 hours, in the polymer capsules formed withRL39-117, RL39-118, and RL39-115). At present, bovine spermatozoa havebeen maintained over an 18 hour period. At least 70% of the spermatozoadisplayed the typical pattern of activated, progressive, forwardmotility characteristic of viable sperm after being released from thepolymer at 37° C. Successful in vitro fertilization of in vitro maturedbovine oocytes demonstrated that these sperm are in fact, capable offertilization of an occyte.

EXAMPLE 4 Sperm Mortality as Function of Retention Time

Bovine sperm obtained from a "swimup column" were diluted with polymerto a final density of approximately 6000/ml in a rectangular mold 10mm×25 .nm×5 mm. The liquid polymer was gelled in an autmosphere of 90%N₂, 5% CO₂ and 5% O₂ with the temperature brought to 7° C. over a periodof 5 hours. The molds were kept at refrigerator temperatures for as manyas 14 days, after which time, they were placed on the stage of anincubator enclosed microscope at 10° C. The temperature was increasedgradually to 37° C. over a period of 3 hours. Throughout this periodcontinuous time-lapse video recording was accomplished. The first signsof sperm motility were observed at 8 hours when tail movement becameapparent. By 10 hours vigorous tail motion was observed and while thepolymer was still in semigel state, sperm head motion was apparent. By12 hours approximately 40% of the sperm showed complete motility with atleast half presenting progressive forward activated motility. By 14hours the polymer was completely liquified and sperm motion essentiallynormal. Loss of spermatozoa (nonmotile; abnormal gross morphology) wasapproximately 25-30%. In some experiments, sperm loss ranged from a lowof 20% to a high of 71%. The typical incidence of sperm loss as afunction of time in polymer is shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        SPERM MORTALITY AS A FUNCTION                                                 OF TIME IN POLYMER                                                            TIME (DAYS)    % VIABLE                                                       ______________________________________                                        1              85                                                             2              85                                                             3              85                                                             5              85                                                             8              71                                                             10             50                                                             14             48                                                             ______________________________________                                    

EXAMPLE 5 Effect of Polymer Concentration

Another important series of experiments demonstrates the criticalassociation between polymer concentration and retention of viability.With small incremental increases in polymer concentration, viability, asjudged from sperm morphology and motility patterns after release frompolymer, decreases dramatically. The results of a typical experiment inthis series, utilizing polymer RL39-110, are presented in Table 5.

                  TABLE 5                                                         ______________________________________                                        CORRELATION BETWEEN POLYMER                                                   CONCENTRATION AND SPERM VIABILITY                                             CONCENTRATION (%)  % VIABLE*                                                  ______________________________________                                        1                  75                                                         1.5                75                                                         3.5                68                                                         4.0                50                                                         5.0                42                                                         6.5                20                                                         8.5                 0                                                         ______________________________________                                         *after 4 days in polymer at 7C with 18 hr warm period                    

These results demonstrate the critical nature of polymer concentrationand formulation for successful preservation of spermatozoa. For example,similar studies showed that polymers RL39-110 and 111 were well suitedfor preservation of mouse and human sperm, but not for bovine. Thispoints out the importance of testing various polymer formations astaught above in optimizing the capsule for a new species.

EXAMPLE 6 Effect of Heating and Cooling Rates

In another series of polymer experiments, the correlation between ratesof cooling and heating on sperm viability was determined. All assayswere performed with sperm entrapped in polymer for 5 days at 7° C. Theinfluence of cooling rate is shown in Table 6 and heating in Table 7.

                  TABLE 6                                                         ______________________________________                                        SPERM VIABILITY AS A FUNCTION                                                 OF COOLING RATE                                                               TIME (HRS) REQUIRED TO COOL TO 7C                                                                      % VIABILITY                                          ______________________________________                                        3                        24                                                   5                        32                                                   8                        59                                                   12                       60                                                   16                       74                                                   ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        SPERM VIABILITY AS A FUNCTION                                                 OF RATE OF HEATING                                                            TIME (HRS) TO 37C % VIABILITY                                                 ______________________________________                                         3                 0                                                           6                27                                                          10                42                                                          14                55                                                          16                79                                                          18                80                                                          ______________________________________                                    

These experiments utilized formulation RL39-114 at 4.5% concentration.

EXAMPLE 7 Preparation of a typical polyurethane-polyether polymer(RL39-118)

The polyether moiety was prepared by mixing 136 grams of polyoxyethyleneglycol (Carbowax 8,000) and 3.8 grams of diethylene glycol with stirringat 77° C. to form a homogeneous melt. The temperature was allowed todecrease to 65° C. While continuing the stirring, 16.63 grams ofmethylene biscyclohexyl-4,4'diisocyanate were added. When thetemperature decreased to 60° C., 0.15 cc of dibutyl tin dilaurate (T12)was added and the mixture allowed to heat up to about 70° C. Thereaction mass was then poured into a polypropylene pan. Upon completionof the pouring operation, the pan was placed in an air circulating ovenat 100° C. and maintained in the oven for one hour to cure the polymer.

After cooling the mass to ambient room temperature, the polymer mass wascut into small pieces. A sufficient amount of the small pieces was mixedwith water to form a mixture containing 2% to 5% solids. The mixture wasstirred while increasing the temperature of the mixture to 95° C.wherein the polymer comes out of solution and agglomerates. Thetemperature is then reduced with continuous mixing to produce a shearingaction; preferably by hand.

As the solution cools to about 70°-75° C. it begins to become clearer asthe polymer begins to redissolve. As the temperature continues to fall,the solution increases in viscosity and becomes almost transparent. Thecooling is stopped near 30° C. and the solution gel. If this has notproduced a gel that is homogeneous, the cycle may be repeated.

The softening point and rigidity of each type of gel can be altered bychanging the precent of solids of the resin. Higher resin contentproduced more rigid and higher softening point gels.

The gel is then made into a dry form which is reconstituted with culturemedium. First the gel is spread out on a suitable nonstick substratesuch as polypropylene or polyethylene to a thickness of about 1/16". Thegel is then dried to produce a thin pliable film. This film is easilycut or torn into small pieces, or can be prepared in a powder form. Thedrying can take place in a vacuum oven at room temperature, acirculating oven at or below 50° C., or in a freeze dryer. The resultingdry polymer film is then cut into small pieces and warm water at about35°-45° C. is added and stirred for about five minutes or until all thepolymer is in solution to produce the desired solids content.

The gel will reach its final rigidity at room temperature in about onehour. Air bubbles can be rapidly removed from the gel by centrifugationor storing in a closed container at 40° C. for 6-12 hours. After severalwashes with deionized H₂ O to remove any toxic substances that may havebeen produced during the preparation of the polymer, the dried polymercan be reconstituted with any culture media that will supportspermatozoa in vitro.

A hydrogel containing 5% of the above described polymer with an aqueousculture medium is a flowable, somewhat syrupy liquid at 37° C. but whencooled to room temperature (20°-22° C.) sets into a solid gel. Theliquid hydrogel may be brought to the desired pH for a specific sperm bythe addition of a buffer solution or a dilute acid or alkaline solutionas may be appropriate.

RL39-117 is prepared identically except for the use of 76.5 g ofCarbowax 4500.

I claim:
 1. A method of encapsulating viable .[.mammalian.]..Iadd.vertebrate animal .Iaddend.sperm for storage or transfer, andsubsequent insemination of a female .[.mammal.]. .Iadd.vertebrateanimal.Iaddend., which comprises encapsulating the sperm with a nontoxichydrophilic polymer, said polymer being essentially freely flowable atthe .Iadd.normal .Iaddend.temperature of the .[.uterus.]..Iadd.reproductive tract .Iaddend.of such .[.mammal.]. .Iadd.vertebrateanimal, which temperature is substantially greater than 22° C.,.Iaddend.the polymer at the latter temperature being permeable tomolecules of a predetermined size but not to the sperm whereby the spermmay receive nutrients and excrete waste products through themicrocapsule without escaping; the sperm escaping from the microcapsuleat said .[.uterine.]. .Iadd.reproductive tract .Iaddend.temperature whenthe polymer becomes freely flowable.
 2. The method of claim 1, whereinthe transition of the polymer to the liquid state is completed within 14hours after it is brought to 37° C.
 3. The method of claim 1, whereinthe sperm are encapsulated by (a) providing viable sperm suspended in asuitable culture medium, (b) adding the polymer to the said spermsuspension, transferring the mixture into molds corresponding to thedesired dimensions of the microcapsules, and (c) cooling the mixtureuntil it solidifies into a non-free flowing gel.
 4. A method ofinseminating .[.an.]. .Iadd.a vertebrate animal whose normalreproductive tract temperature is substantially greater than22°.Iaddend., which comprises inseminating a receptive female of aspecies of .Iadd.said .Iaddend.animal with the encapsulated sperm ofclaim 1, .[.said female.]. .Iadd.and .Iaddend.permitting theencapsulated sperm to warm to the temperature of the .[.uterus.]..Iadd.reproductive tract .Iaddend.of the female, at which temperaturethe capsule is .[.liquiified.]. .Iadd.liquified .Iaddend.and the spermreleased.
 5. The method of claim 4 in which the sperm are first held ata temperature at which the capsule is essentially solid.
 6. An articleof manufacture comprising viable sperm of a .[.mammalian.]..Iadd.vertebrate animal .Iaddend. species .Iadd.whose normalreproductive tract temperature is substantially greater than 22° C.,.Iaddend.encapsulated in a microcapsule formed by a nontoxic hydrophilicpolymer which is essentially solid at 4° C., said sperm being releasedby liquefaction of said polymer at the temperature of the .[.uterus.]..Iadd.reproductive tract .Iaddend.of females of such species, said spermremaining capable of fertilizing such females after such encapsulationand release.
 7. The article of claim .[.4.]. .Iadd.6.Iaddend., whereinthe polymer is a polyurethane polymer.
 8. The .[.method.]. .Iadd.article.Iaddend.of claim 7, wherein the polymer is selected from the groupconsisting of polyurethane polyoxyethylene and polyurethane polyesterpolymers.
 9. The article of claim .[.4.]. .Iadd.6 .Iaddend.in which saidmicrocapsule encloses an atmosphere which is about 5% O₂, 5% CO₂ and 90%N₂.
 10. The article of claim .[.4.]. .Iadd.6 .Iaddend.in which thepolymer is selected from the group consisting of RL39-115, RL39-117, andRL39-118.
 11. The article of claim .[.4.]. .Iadd.6 .Iaddend.in which thepolymer forms a microcapsule enclosing a culture medium for the sperm,said medium having a pH of about 7.37-7.42.
 12. The article of claim.[.4.]. .Iadd.6 .Iaddend.in which the polymer forms a microcapsuleenclosing a culture medium for the sperm, said medium provding calciumlactate in an amount sufficient to render the sperm hypermotile whenunrestrained by the microcapsule.
 13. The article of claim .[.4.]..Iadd.6 .Iaddend.where the polymer forms a translucent microcapsuleabout the sperm, whereby its motility may be observed.
 14. The articleof claim 6 wherein the sperm are held in a state of reduced motility andmetabolic activity prior to liquefaction, the motility and metabolicactivity of the sperm being restored after liquefaction.
 15. The articleof claim 6, where more than 50% of the sperm remain viable for 14 daysafter encapsulation and storage at 4° C. .Iadd.
 16. The method of claim1 in which the animal is a mammal. .Iaddend. .Iadd.17. The method ofclaim 4 in which the animal is a mammal. .Iaddend. .Iadd.18. The articleof claim 6 in which the animal is a mammal. .Iaddend. .Iadd.19. Themethod of claim 1 in which the animal has a normal reproductive tracttemperature of at least about 37° C. .Iaddend. .Iadd.20. The method ofclaim 4 in which the animal has a normal reproductive tract temperatureof at least about 37° C. .Iaddend. .Iadd.21. The article of claim 6 inwhich the animal has a normal reproductive tract temperature of at leastabout 37° C. .Iaddend. .Iadd.22. The method of claim 19 in whic theanimal has a normal reproductive tract temperature of about 37° C..Iaddend. .Iadd.23. The method of claim 20 in which the animal has anormal reproductive tract temperature of about 37° C. .Iaddend..Iadd.24. The article of claim 21 in which the animal has a normalreproductive tract temperature of about 37° C. .Iaddend.